Wild mouse species and the various inbred laboratory mouse strains differ from one another in their susceptibility to the mouse gammaretroviruses and retrovirus-induced cancers. These differences are due to variations in specific host genes, and we have been engaged in an ongoing effort to identify and characterize several mouse genes involved in virus resistance. Our major interest has been focused on factors that interfere directly with virus infection and replication, and we focus our efforts on those factors that inhibit virus entry and the early post-entry stages of the virus replicative cycle. At the level of entry, there are two types of resistance genes that target the receptor-virus interaction. Receptors can be blocked by virus envelope glycoprotein produced by endogenous retroviruses, or resistance can be caused by polymorphisms in the cell surface receptor. After the gammaretrovirus enters the receptive cell, reverse transcription and translocation to the nucleus can be inhibited or altered by virus resistance factors Fv1, mApobec/Rfv3, and TRIM5alpha. Our current aim is to characterize these resistance factors and the viruses they target, define the origin and extent of antiviral activity in Mus evolution, and elucidate the responsible mechanisms. This work relies heavily on wild mice because laboratory strains provide only a limited sampling of the genetic diversity in Mus. Also, wild mouse species allow us to examine survival strategies in natural populations that harbor virus and to follow the evolution of the resistance genes. These mice additionally provide a source of novel resistance genes and virus variants. One set of projects aims to identify viral and cell receptor determinants responsible for virus binding and entry. We are currently working on the XPR1 receptor for the xenotropic/polytropic mouse gammaretroviruses (XP-MLVs). We have determined that, in mouse populations exposed to infectious virus, virus resistance is mediated by polymorphisms of the cell surface receptor. We have now identified a total of four XPR1 susceptibility variants in wild mice and described the geographic and species distribution of the Mus Xpr1 variants. Three of the four receptors restrict entry by two or more of the virus host range variants that rely on XPR1, and all three of these receptors evolved in populations exposed to X-MLVs. Because XPR1 homologues are found in all eukaryotes, and because all mammals but the laboratory mouse have X-MLV susceptible receptors, we looked for naturally occurring restrictive receptors in a related set of species outside the mammalian lineage. We found restrictive receptors in chickens as well as in other fowl and raptor species, and demonstrated that the gene is under positive selection which is evidence of genetic conflicts that can result from antagonistic interactions with pathogens. All of the birds with disabling mutations are native to areas of Asia populated by the virus-infected mouse species M. m. castaneus, suggesting that contact between these species favored selection of mouse virus-resistant receptors in the birds. In another series of experiments, we have been using phylogenetic methods to identify and characterize host genes that have had an anti-viral role in the genus Mus. Among the mouse genes responsible for resistance to mouse leukemia viruses is the mouse APOBEC (mA3) gene, a cytidine deaminase gene known to restrict other retroviruses in mice and in humans. In previous studies we discovered that the mA3 allele in virus resistant mice is disrupted by insertion of the regulatory signals of a mouse leukmia virus that correlated with enhanced mA3 expression in virus resistant mouse strains and species. We also identified sites in mA3 under positive selection that specify residues in two loops along the groove that forms the mA3 active site. More recently we focused on the fact that mA3 transcripts have different splicing patterns in virus-resistant mice like C57BL/6 compared with virus-sensitive strains like BALB/c. C57BL/6 transcripts lack exon 5; this exon is spliced into mA3 of BALB/c and separates the two cytidine deaminase domains. We have now showed that mA3 exon 5 is a functional element that influences protein synthesis at a post-transcriptional level. We also used in vitro splicing assays to identify two critical polymorphisms affecting the inclusion of exon 5 into mA3 transcripts: the number of TCCT repeats upstream of exon 5 and a single nucleotide substitition within exon 5. We also found that distribution of exon 5 into mA3 mRNA is a relatively recent event in the evolution of mice. The widespread geographic distribution of the exon 5-including genetic variant suggests that in some Mus populations the cost of maintaining an effective but mutagenic enzyme may outweigh its antiviral function. One of the unique features of gammaretroviruses is that they contain an additional extended form of the gag polyprotein, termed glycogag. This glycoprotein is thought to promote virus replication and have a role in disease progression. We made use of a recently isolated gammaretrovirus, termed XMRV, which is the only known virus that lacks the consensus glycogag sequence. We did functional assays to confirm that this virus lacks glycogag function, and we analyzed the endogenous copies of the gammaretroviruses in the sequenced genome for glycogag. We found that glycogag was missing from two subgroups of these leukemia viruses, indicating that loss of this sequence is a relatively recent event with limited subspecies distribution. Few mouse leukemia viruses are cytopathic, and these few viruses have restricted host range. We forced passaged one of these cytopathic viruses in restrictive cells and isolated a novel variant that has broad mouse-tropic host range, and is cytopathic in all mouse cells. We identified a single second site mutation in the envelope glycoprotein of this virus responsible for its expanded host range. This site is not positioned within the receptor binding pocket of the envelope glycoprotein, but is at the apex of this glycoprotein, where it is likely to have secondary contacts with the receptor.